HIV resistance to clinically approved therapeutics is an increasingly serious clinical problem. New therapeutics are needed, especially those against unaddressed HIV targets, such as RT-associated ribonuclease H (RNH). This project will assist in the validation and preclinical development of RNH inhibitors (RNHIs) prepared by semi-synthetic optimization of plant cell culture derived natural products. The aims of this proposed project are 1. to conduct detailed biochemical and virologic characterizations (mechanism of action) of a series of novel RNHIs being pursued by scientists at the partnering company, Millenia Hope. The current leads have sub-micromolar inhibitory activity against RNH in vitro and against HIV replication in PBMC;2. to screen a library of 150,000 plant-derived partially purified and purified natural products for additional RNHIs. Hits will be validated in a variety of secondary assays and promising leads will undergo optimizations in other project components;and 3. to characterize in detail the validated hits obtained in the screening program. The work in this project is an essential component of the overall preclinical development program, and is intimately associated with that of the others (Project 1: Isolation &Optimization, and Project 3: Structural and Computational Biology) in the overall iterative multi-project program. The goal of the entire multi-application program is to develop 2-3 first generation leads and 4-6 back-up leads with low nM potencies. RNH is essential for HIV replication, yet there are no drugs directed at this target. This research program will develop a first-in-class therapeutic agent targeting RNH for use in the treatment of HIV infection. As RNH is a novel target unaddressed by currently used HIV drugs, it is likely that RNHIs developed in this program will be useful for the treatment of drug-resistant HIV.